Recent studies have shown that the cardiac b2-adrenoceptor (b2-AR) contractile response is mediated by a localized cAMP/protein kinase (PKA)-dependent augmentation of L-type Ca2+ current (ICa) and occurs in the absence of PKA-mediated phosphorylation of more remote proteins, e.g. phospholamban (PLB), an important modulator of cardiac relaxation. Since b2-AR is also coupled to Gi proteins, and pertussis toxin (PTX) augments the contractile response following b2-AR stimulation, we hypothesized that Gi might be involved in the localization of b2-AR signaling. Thus, we examined the effect of PTX on the kinetics of b2-AR mediated site-specific PLB phosphorylation and relaxation in rat cardiomyocytes. PTX enhanced the effect of the b2-AR agonist zinterol (Zin, 10-5 M) on contraction amplitude and rescued its relaxation effect, whereas the b1-AR mediated response remained unchanged. Zin induced a cAMP-dependent PLB phosphorylation in a time and dose-dependent manner (EC50=48.6+/-1.8 nM) only in PTX-treated cardiomyocytes, with a maximal increase in phosphorylation by 6.5-fold, similar to that induced by the b1-AR agonist norepinephrine (37.9+/-4.7 and 36.7+/-7.7, respectively). Therefore, the coupling of b2-AR to Gi proteins is a key factor for the localization of b2-AR signaling to a subsarcolemmal domain and this constraint can be removed by PTX.